Ouhenach et al. BMC Medical Genetics(2020) 21:240
https://doi.org/10.1186/s12881-020-01167-y

RESEARCH ARTICLE

Open Access

Application of next generation sequencing
in genetic counseling a case of a couple atrisk of cystinosis
Mouna Ouhenach1,2* , Abdelali Zrhidri1,2, Imane Cherkaoui Jaouad1,2, Wiam Smaili1 and Abdelaziz Sefiani1,2

Abstract
Background: In Morocco, consanguinity rate is very high; which lead to an increase in the birth prevalence ofinfants with autosomal recessive disorders. Previously, it was difficult to diagnose rare autosomal recessive diseases.
Next Generation Sequencing (NGS) techniques have considerably improved clinical diagnostics. A genetic diagnosis
showing biallelic causative mutations is the requirement for targeted carrier testing in parents, prenatal andpreimplantation genetic diagnosis in further pregnancies, and also for targeted premarital testing in future couples
at risk of producing affected children by a known autosomal recessive disease.
Methods: In this report, we present our strategy to advise a future couple of first cousins, whose descendants
would risk cystinosis; an autosomal recessive lysosomal disease caused by mutations in the CTNS gene. Indeed, ourfuture husband’s sister is clinically and biochemically diagnosed with cystinosis in early childhood. First, we opted
to identify the patient’s CTNS gene abnormality by using (NGS), then we searched for heterozygosity in the couple’s
DNA, which allows us to predict the exact risk of this familial disease in the future couple’s offspring.
Results: We have shown that the future husband, brother of the patient is heterozygous for the familial mutation.
On the other hand, his future wife did not inherit the familial mutation. Therefore, genetic counseling was
reassuring for the risk of familial cystinosis in this couple’s offspring.
Conclusions: We report in this study, one of the major applications of (NGS), an effective tool to improve clinicaldiagnosis and to provide the possibility of targeted premarital carrier testing in couples at risk.
Keywords: Next generation sequencing, Consanguinity, Genetic counseling, Cystinosis

Background
Morocco is one of the countries with a high rate of consanguinity in the Mediterranean, where consanguineous
marriages account for 15.25% of all marriages, and 58.46%of them are between first cousins [1]. Autosomal recessive
disorders are strongly associated with consanguineous
marriages; the frequency of these marriages are estimated* Correspondence: modruna@gmail.com
1
Department of Medical Genetics, National Institute of Health, 27, Avenue
Ibn Battouta, BP 769, Rabat, Morocco2
Human Genomics Center, Faculty of Medicine and Pharmacy, Rabat,
Moroccoat 59.09% among Moroccan families with autosomal recessive disorders [1].
Previously, the large number of potential genes and thephenotypic variability associated with many known genetic causes, have made the diagnosis of rare autosomal recessive disorders very difficult. In recent years, significant
advances are achieved using next generation sequencing(NGS) technology in medical practice especially in the
diagnosis of children with autosomal recessive diseases.
Indeed, NGS has significantly improved clinical diagnostic
compared to traditional sequencing methods. Thus, so an
accurate diagnosis will enable more appropriate genetic© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Ouhenach et al. BMC Medical Genetics(2020) 21:240

counseling and targeted parental carrier testing, prenatal
and preimplantation genetic diagnosis in subsequent pregnancies, and targeted premarital genetic testing in at-risk
couples.
Here we report our experience with targeted premarital testing in a first-degree consanguineous couple, whohave a family history of a severe form of cystinosis.
Cystinosis is a rare autosomal recessive lysosomal storage
disorder, described in 1903 as a familial cystine accumulation [2], leading to cellular dysfunction, it’s a systemic
disease whereas the kidney involvement is the most serious
clinical event, driving before the age of 20 to end-stage
renal disease in more than 90% of patients [3, 4].
Although cystinosis is a monogenic disease, caused bymutations in the CTNS gene, a large gene of 12 exons
distributed across approximately 23 kb of genomic DNA,
encodes the protein cystinosin and maps to chromosome
17p13. Depending on the severity of mutations affecting
the CTNS gene, there is three major clinical presentations of the cystinosis disease: the infantile nephropathic
form (MIM: 219800), the juvenile nephropathic form
(MIM: 219900), and the ocular non-nephropathic form
(MIM: 219750) [4–6].

Methods
Patients

A Moroccan couple of first-degree cousins intending
marrying (Individuals V-1 and V-2; Fig. 1) were referredto our specialized in genetic counseling clinic. The future husband has a sibling affected by cystinosis with an
early renal failure. Therefore, the couple worried about

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their offspring and came to our center for genetic counseling. Accordingly, we informed the couple about theincreased risk of having children with cystinosis then we
proposed them more precise genetic counseling.
The patient, the future husband’s sister (patient V-3;
Fig. 1), is a 22-year-old girl, diagnosed with cystinosis inearly childhood. Actually, she has a severe form of cystinosis, which progressed towards renal failure at the ageof 6 years old.
Molecular studies

In a first step, we chose to identify the CTNS genepathogenic mutation of cystinosis in the patient (V-3;
Fig. 1). As cystinosis is a monogenic disease due toabnormalities in CTNS gene, which is a large gene of 12exons, we chose to perform a target next-generation sequencing using a gene panel of hereditary diseases (Ion
AmpliSeq™ Inherited Disease Panel), which include
CTNS gene, then we confirmed the patient’s mutation.
In a second step, we screened for heterozygosity of thefamilial mutation in our couple. Consequently, we wereable to predict the exact risk of this familial disease in
their offspring.
Written informed consent was obtained from the patient and the future couple prior to the implementationof the genetic study reported here.
Detection of the familial Cystinosis causative mutation-DNA and NGS

Prior to perform the genetic study reported here, written
informed consent was obtained from the patient and theFig. 1 Pedigree of the studied couple (indicated with asterisks). Affected patient with cystinosis is shown by black circle (In the chapter “Patients”)

Ouhenach et al. BMC Medical Genetics(2020) 21:240

future couple (patient V-3 and the individuals V-1 andV-2; Fig. 1). we Used Invitrogen kit (PureLink™ Rapid
Gel Extraction and PCR Purification Combination Kit/
K220001) to extract genomic DNA from peripheral
blood lymphocytes, and then we used the Qubit dsDNA
HS (High Sensitivity) Assay Kit to quantify DNA with
Qubit® Fluorometer (Invitrogen™), according to the manufacturer’s instructions.
According to the NCBI ClinVar database, the Ion
AmpliSeq™ Inherited Disease Panel can provide a highdegree of multiplexed target selection of all exons andsome gene introns associated with more than 700
unique genetic diseases. In our case, we analyzed all coding exons of the CNTS gene. Libraries were preparedusing Ion AmpliSeq Library Kit v2.0 (Life Technologies,
Carlsbad, CA, USA) for this patient and for two other
patients affected by other hereditary diseases. The panel
contains three primer pools. For amplification, 2 μL of
5× Ion Ampliseq HiFi Mix (Thermo Fisher Scientific),
2 μL of 5× Ion Ampliseq™ inherited disease pool, 10 ng
of genomic DNA per reaction, and maked up to a final
volume of 10 μL with nuclease-free water. The final
10 μL polymerase chain reaction (PCR) mixture was applied with a temperature profile of 99 °C for 2 min, 99 °C
for 15 s, and 60 °C for 8 min for 14 cycles, and finallykept at 10 °C. The primer sequences were partiallydigested. Adapters and one of 16 barcodes of the Ion
Xpress Barcode Adapters 1 to 16 Kit (Thermo Fisher
Scientific Life Sciences Solutions, Carlsbad, CA, USA),was applied to each sample. The library was quantified
using Qubit dsDNA HS analysis kit (Molecular Probe,
Eugene, OR, USA) on Qubit 2.0 fluorometer, and an
equimolar amount of each library is used to prepare
templates for clonal amplification. Emulsion PCR was
performed on the OneTouch2 system (Life Technology
in Carlsbad, California, USA) using the Ion PGM HI-Q
OT2 kit (Life Technology in Carlsbad, California, USA).
The template was enriched using Ion OneTouch ES(Carlsbad, California, U.S. Life Technology Company),
and Ion 318 Chip v2 was prepared for loading (Carlsbad,
California, U.S. Life Technology Company). According
to the manufacturer’s instructions, Ion PGM HI-Q
sequencing was used to perform sequencing runs on the
Ion Torrent Personal Genome Machine (PGM, Life
Technologies).
The Torrent Mapping Alignment Program aligner implemented in Torrent Suite software (Thermo Fisher
Scientific) v5.4 was used to compare the raw sequence
data in FASTQ format with the hg19 human reference
genome. For SNV calling, we used plug-in Torrent Variant Caller v5.2.0.34 (Thermo Fisher Scientific) to set up
a variant call format file. Then we used a default settingof germline low-stringency parameters: the minimal variant frequency is 0.1, the minimum variant quality is 10,

Page 3 of 6

the minimum coverage is 5 ×, the maximum strand bias
is 0.98, and the minimum variant score is 10, for Torrent
Variant Caller analysis. Thus, the candidate variants
were only obtained when variant frequency at a given
position is ≥20% and variant coverage is ≥20×. IGV (version 2.1.16) was used to view the mapping and annotationof the sequence on the graphical interface.
Reported variants are confirmed in the Human Gene
Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/
ac/index.php), Clinvar and previous publications.
The sequence data analysis detected a potential deletion
spanning exons 4 and 5 in the CTNS gene.
-RNA and RT- PCR

Initially, Total RNA was isolated from leukocytes using
an RNA extraction kit (RNeasy Mini Kit, Qiagen GmbH,
Hiden, Germany), and then cDNA was performed using
Superscript III Reverse transcriptase (Life Technologies)
according to the manufacturer’s protocol. Thus, cDNA
amplification of the CTNS gene of the patient (V-3;
Fig. 1), was performed to confirm the deletion (a potential deletion spanning exon 4 and 5), using the primers[forward: ACTGGGCGAAGGGAGGACT and reverse
CACTCCAGGAGGCACCACA]. The reaction products
were analyzed by 1% agarose gel electrophoresis. Then,
Sanger sequencing was done using dye terminator chemistry (ABI Prism BigDye v3.1) and run on automated sequencer Applied Biosystems Prism 3130 DNA Analyzer.
Determination of genetic profile of the couple at risk

In order to determine our couple’s (Individuals V-1, V-2;
Fig. 1) genetic profile for the familial mutation, we used
RT-PCR, the same method as above. Amplified products
were also analyzed on a 1% agarose gel electrophoresis.
Then Sanger sequencing is done using dye terminatorchemical reagents (ABI Prism BigDye v3.1) and runs on
the automated sequencer Applied Biosystems Prism
3130 DNA Analyzer.

Results
Detection of the familial Cystinosis causative mutation

To confirm the diagnosis of cystinosis at the molecularlevel, we carried out a mutational analysis as describedin the patients and methods section. As follows, we used
targeted next generation sequencing to detect mutationsin the promoter and exons of the CTNS gene in our
cystinosis affected family member (patient V-3; Fig. 1).
The sequence data analysis detected a potential deletion
spanning exons 4 and 5 (no reads are generated forthese tow exons (Fig. 2a)) and no other pathogenic variant is detected in CTNS gene. Then, we performed RTPCR and cDNA Sanger sequencing, which confirmed
the NGS suspected deletion (Fig. 2b). This result allows
us to identify the causal mutation of cystinosis in thisOuhenach et al. BMC Medical Genetics(2020) 21:240

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Fig. 2 (a) The IGV image showing a deletion of two exons 4 and 5 in the CTNS gene detected by the Ion Torrent next-generation sequencing
platform. (b) Sequencing diagram of cDNA showing the homozygous deletion of the two exons 4 and 5 in CTNS gene (1) in compare to an
normal sequence (2). (c) Image of agarose gel after RT-PCR amplification products electrophoresis. (V; 3): the RT-PCR amplified products gel
analysis showing the presence of a small fragment comparing to those of a normal sample. (NC): the RT-PCR reaction products gel analysis of anormal sample. (V; 2): Agarose gel analysis of the RT-PCR amplified products of the future husband, which showed the presence of two
fragments, one fragment has the same size as those of the control sample and the other fragment has the same size of the patient’s sample. (V;1): The Agarose gel analysis of the future wife, showing the presence of one fragment of the amplified target cDNA who have the same size as
those of the control sample. (N): Negative control. (In the chapter “Results”)

family: homozygous deletions carrying both exons 4 and5 [c.(61 + 1_62–1)_(225 + 1_226–1); NM_004937.3] of
the CTNS gene, according to the HGVS format (Fig. 2a).
Determination of genetic profile of the couple at risk

In order to determine our couple’s genetic profile, we used
the RT-PCR in three individuals (the couple and theaffected patient (Fig. 1: Individuals V-1, V-2, and the patient)). The agarose gel analysis of the patient’s RT-PCR
reaction product (V-3; Fig. 2c) shows that the amplified
cDNA fragments are smaller than those of the control
sample. We compared this result to our couples RT-PCR
products: The future husband’s agarose gel analysis of the
RT-PCR reaction products showed the presence of two
fragments (V-2; Fig. 2c), one fragment has the same sizeas those of the control sample, and the other fragment has

the same size as the amplified fragment of the affected patient. Therefore, the future husband has two alleles, one
allele carrying the deletion, and one normal allele, so he
is heterozygous for the familial mutation of cystinosis.
Regarding the future wife (V-1; Fig. 2c), agarose gelanalysis revealed the presence of one amplified target
cDNA fragment at the same size as those of the control
sample (V-1; Fig. 2c). Accordingly, the future wife ishomozygous normal, so she is not a carrier of the familial mutation.

The genetic counseling

Therefore, genetic counseling is reassuring for the risk ofcystinosis due to the familial mutation in their offspring.
However, the couple remains at risk due to inbreeding.

Ouhenach et al. BMC Medical Genetics(2020) 21:240

Discussion
We are describing a family with an autosomal recessive
disease called cystinosis, and we presented our strategy
to advise a consanguineous future couple of first-degree
cousins (Individuals V-1 and V-2; Fig. 1), whose descendants would be at risk of this disease. The awareness of
the risk of consanguineous marriages is getting higher inour population; we receive in our Department ofMedical Genetics, National Institute of Health, Rabat,
Morocco, many cases of genetic counseling of consanguineous families and couples concerned about theirdescendants. The aim of this study is to report one themajor application of the Next-generation sequencing(NGS) technology in genetic counseling.
This couple (Individuals V-1 and V-2; Fig. 1) that we
received in our consultation, were worried about theiroffspring, they are a first-degree consanguineous couple,
who have a family member affected with severe infantile
cystinosis (MIM: 219800). Our genetic counseling strategy is to perform an accurate molecular diagnosis of
cystinosis disease in this family and then determine thegenetic profile of the familial mutation in our couple.
Cystinosis disease is a rare lysosomal storage disease; it
is a systemic disease whereas kidney involvement is themost serious clinical event. The prevalence of cystinosis
disease is known in some populations, it varies between
1/192,000 in Australia to 1/26,000 in France [7, 8]. However, the incidence of cystinosis disease is still lacking in
the North African population.
Cystinosis disease is due to biallelic mutations in the
CTNS gene (17p13.2), which is a gene of 12 exons cystinosin. A large number of mutations are found in CTNS

Page 5 of 6gene, about 168 reported in the Human Gene Mutation
Database at the Institute of Medical Genetics in Cardiff(HGMD, http://www.hgmd.cf.ac.uk/ac/gene.php?gene=
CTNS). The most common pathogenic mutation is a 57kb deletion, which represent nearly 50% of CTNS mutant alleles in patients of North European and North
American origin [9, 10], but no recurrent mutation has
been found in the north African or middle eastern
population.
In our case, due to the large large number of CTNS
gene exons, we used the next generation sequencing tocarry out a molecular diagnosis of cystinosis disease inthis family. Furthermore, cystinosis disease is due tobiallelic pathogenic mutation, so even a large deletion
can also be suspect by this technology.
Effectively, the analysis of our patient (V-3; Fig. 1),
identified a deletion spanning exons 4 and 5 of CTNS
gene ((a); Fig. 2). We confirmed this mutation [c.(61 + 1_
62–1)_(225 + 1_226–1), NM_004937.3] by RT-PCR ((c);
Fig. 2) and cDNA Sanger sequencing ((b); Fig. 2). Thisdeletion leads to a short mRNA, resulting in a frameshift
and truncated version of CTNS protein (Fig. 3), and was
already reported as pathogenic [11]. That explains thechildhood-onset and the severe form of our patient’s
phenotype. Therefore, we conclude that this allele variant should be classified as pathogenic and there is noneed to screen it in a healthy control.
Next generation sequencing technology has helped us
to obtain a more rapidly, and more accurate diagnosis.
NGS is potentially allowing for more efficient genetic
counseling, premarital targeted testing in the couple atrisk, and did helped us to predict the exact risk of thisFig. 3 Schematic representation of the Exon 4 and 5 deletion of CTNS gene and their consequence in cDNA of this region of the gene.
(In the chapter “Discussion”)

Ouhenach et al. BMC Medical Genetics(2020) 21:240

familial disease in this couple’s offspring; therefore, wewere able in this case to reassure the couple about therisk of cystinosis in their descendent. However, thecouple remains at risk for other recessive diseases due tothe inbreeding.

Page 6 of 6Consent for publication
Written informed consent was obtained from the patient and the future
couple for the publication of this report.
Competing interests
The authors declare that they have no competing interests.
Received: 6 August 2019 Accepted: 10 November 2020

Conclusions
Here we reported one of the major applications of next
generation sequencing. These tests become more accessible and faster, it would be more cost-benefit for patientsand their families. Our perspective is to create a screening test for the most common autosomal recessive disease in our population, as a health public strategy inorder to reduce the prevalence of these diseases.
Abbreviations
NGS: Next Generation Sequencing; PGM: Personnel Genome Machine;
DNA: Deoxyribonucleic acid; PCR: Polymerase chain reaction;
cDNA: Complementary Deoxyribonucleic Acid; RT-PCR: Reverse transcription
polymerase chain reaction
Acknowledgements
The authors would like to thank the patients and their family. We are
grateful to all of the staff of the Department of Medical Genetics of the
National Institute of Health for their continuous support.
Authors’ contributions
MO carried out the genetic counseling strategy, the molecular study, andparticipated in the writing of the manuscript. AZ and ICJ participated in thedesign, the molecular study, and drafted the manuscript. WS, performed
clinical examinations, literature search, provided samples, and participate inmanuscript preparation. AS conceived the study, participated in its design
coordination and helped to edit the final version.. All authors read andapproved the final manuscript.

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Publisher’s Note
Funding
No external funding was available for the study.
Availability of data and materials
Web links and the full names of the data banks/repositories corresponding
to all of the datasets obtained from web-based sources and subsequently
analysed in this study are cited below:-human genome 19 reference:https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.13/- NM_004937.3:
http://www.ncbi.nlm.nih.gov/nuccore/NM_004937.3- Human Gene Mutation Database:
http://www.hgmd.cf.ac.uk/ac/index.php- List of inherited diseases associated to the Ion AmpliSeq™ Inherited Disease
Panel target gene, according to NCBI ClinVar database, wich include
cystinosis disease:
ftp://ftp.ncbi.nlm.nih.gov/pub/clinvar/gene_condition_source_id
The principal data (causative variant in the patient and, agarose gel analysisof the RT-PCR amplification products of the patient and the couple and Sequencing diagrams of cDNA) generated and/or analyzed during the current
study are included in the published article. The complete datasets used and/or analyzed during the current study are available from the corresponding
author upon request.
Ethics approval and consent to participate
Written informed consent for molecular genetic testing was obtained fromthe patient and the future couple. Statements of their signed consent areavailable onrequest. This study was approved by The Ethics Committee of
the National Instituteof Health in Rabat, Morocco.

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